Synthesis of isoquinuclidine analogs of chloroquine: Antimalarial and antileishmanial activity

Article abstract of DOI:10.1016/j.bmc.2006.11.024

The isoquinuclidine (2-azabicyclo[2.2.2]octane) ring system may be viewed as a semi-rigid boat form of the piperidine ring and, when properly substituted, a scaffold for rigid analogs of biologically active ethanolamines and propanolamines. It is present in natural products (such as ibogaine and dioscorine) that display interesting pharmacological properties. In this study, we have expanded our continuing efforts to incorporate this ring system in numerous pharmacophores, by designing and synthesizing semirigid analogs of the antimalarial drug chloroquine. The analogs were tested in vitro against Plasmodium falciparum strains and Leishmania donovani promastigote cultures. Compounds 6 and 13 displayed potent antimalarial activity against both chloroquine-susceptible D6 and the -resistant W2 strains of P. falciparum. All analogs also demonstrated significant antileishmanial activity with compounds 6 and 13 again being the most potent. The fact that these compounds are active against both chloroquine-resistant and chloroquine-sensitive strains as well as leishmanial cells makes them promising candidates for drug development.

Full text of DOI:10.1016/j.bmc.2006.11.024

Bioorganic & Medicinal Chemistry 15 (2007) 3919–3925
Synthesis of isoquinuclidine analogs of chloroquine: Antimalarial
and antileishmanial activity
M. O. Faruk Khan, Mark S. Levi,à Babu L. Tekwani,§ Norman H. Wilson–
and Ronald F. Borne*
Department of Medicinal Chemistry, University of Mississippi, University, MS 38677, USA
Laboratory for Applied Drug Design and Synthesis, School of Pharmacy, University of Mississippi, University, MS 38677, USA
Received 20 September 2006; revised 8 November 2006; accepted 13 November 2006
Available online 16 November 2006
Abstract—The isoquinuclidine (2-azabicyclo[2.2.2]octane) ring system may be viewed as a semi-rigid boat form of the piperidine ring
and, when properly substituted, a scaffold for rigid analogs of biologically active ethanolamines and propanolamines. It is present in
natural products (such as ibogaine and dioscorine) that display interesting pharmacological properties. In this study, we have
expanded our continuing efforts to incorporate this ring system in numerous pharmacophores, by designing and synthesizing
semirigid analogs of the antimalarial drug chloroquine. The analogs were tested in vitro against Plasmodium falciparum strains
and Leishmania donovani promastigote cultures. Compounds 6 and 13 displayed potent antimalarial activity against both
chloroquine-susceptible D6 and the -resistant W2 strains of P. falciparum. All analogs also demonstrated significant antileishmanial
activity with compounds 6 and 13 again being the most potent. The fact that these compounds are active against both chloroquine-
resistant and chloroquine-sensitive strains as well as leishmanial cells makes them promising candidates for drug development.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
The most important members of these three groups of
alkaloids are the iboga alkaloids.
The isoquinuclidine (2-azabicyclo[2.2.2]octane) ring sys-
tem (1) is present in natural products possessing interest-
ing pharmacological properties such as the alkaloids of
Dioscorea hispida, typified by dioscorine (2), mearsine
(3), a minor alkaloid of the elaeocarpaceous plant Peri-
pentadenia mearsii1 that grows in the rain forests of
north Queensland, and the iboga alkaloids, of which
ibogaine (4) is the prototype. Dioscorine has been
shown to be a toxic central nervous system depressant2,3
and a modulator of the nicotinic acetylcholine receptor.4
HN
1
Ibogaine (4, NIH 10567, EndabuseTM), a natural alkaloid
found in the root, rootbark, stem, and leaves of the Afri-
can shrub Tabernanthe iboga, was isolated over 100
years ago.5–7 Its structure was established 50 years later8
and 10 years later its first total synthesis was reported by
Buchi.9 A number of isoquinuclidine-based pharmaco-
phores prepared over the years have shown interesting
pharmacological activities. Several reviews of the anti-
addictive properties of analogs of the iboga alkaloids
have appeared10–12 as well as a review of iboga alkaloids
and their role as precursors of anti-neoplastic bisindole
Catharanthus alkaloids.13 Recently the synthesis and
biological evaluation of 18-methoxycoronaridine
congeners as potential anti-addictive agents have been
reported.14 More recently, the chemical and biological
properties of isoquinuclidine (4) analogs have also been
reviewed.15
Keywords:
Chloroquine.
Antimalarial;
Antileishmanial;
Isoquinuclidine;
*
Corresponding author. Tel.: +1 (662) 915 5881; fax: +1 (662) 915
5638; e-mail: rborne@olemiss.edu
Present address: College of Pharmacy, Southwest Oklahoma State
University, Weatherford, OK, USA.
à
§
Present address: College of Medicine, University of Arkansas for
Medical Sciences, Little Rock, AR, USA.
National Center for Natural Products Research, University of
Mississippi, Universtiy, MS, USA.
–
School of Biomedical Sciences, University of Edinburgh, Edinburgh,
UK.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.11.024

3920
M. O. Faruk Khan et al. / Bioorg. Med. Chem. 15 (2007) 3919–3925
N-CH3
to yield an amino alcohol intermediate which cyclizes
H3C
H
at very high temperature (ꢀ160 ꢁC). Subsequent hydro-
lysis and Red-AlTM reduction afford the N À ,6-disubsti-
tuted isoquinuclidines.19
CH3
N
H3CO
O
CH3
O
N
H
CH2CH3
N
2
3
O
4
Advantageously, the synthetic route as depicted in
Scheme 1 is employed in the present study. The Diels–
Alder adduct was obtained in high yields using acrylo-
nitrile as the dienophile to furnish nearly equal amounts
of the exo- and endo-products (9) as shown by the NMR
and UV intensity on TLC, the exo isomer being of
slightly higher polarity. The reaction was also performed
using allyl cyanide (vinylacetonitrile) as the dienophile,
but furnished poor yields of the Diels–Alder adduct
(ꢀ20% in total) with a high endo:exo ratio (5:1). This
approach was not optimized further.
Isoquinuclidine is basic, having a pKa of 11, and may be
considered as rigid analog of biologically active ethanol-
amines and propanolamines. This bicyclic amine thus
provides structural similarity with the alkylamino side
chain of several clinically useful drugs. We have recently
expanded our continuing efforts of utilizing this ring sys-
tem in a variety of pharmacophores to the synthesis of
semirigid isoquinuclidine analogs of the widely used
antimalarial chloroquine (6, 7) (Fig. 1). Unfortunately,
resistance to chloroquine has developed thus leading
to its ineffectiveness16 and prompting the need for the
development of new antimalarials.
Scheme 2 is followed in the synthesis of the analogs 11–
15 from isoquinuclidine-6-carbonitrile (10). The amina-
tion of 4-chloroquinoline to give 11 proceeded in fair
yield (ꢀ60%) with a reaction time of 48 h at high tem-
perature (140 ꢁC). Lithium aluminum hydride reduction
of the nitrile group to the amine gave excellent yields
(90%), and dimethylation gave 50% yield. Ethylation
with acetaldehyde produced the monosubstituted sec-
ondary amine 14 as the major product accompanied
by some disubstituted product 15. The products were
purified by column chromatography.
2. Chemistry
Synthetic routes to the isoquinuclidine ring system have
been reviewed recently by Borne and co-workers.15 Use
of (+)-pulegone, in a Mannich-type reaction with acetal-
dehyde and benzylamine, produces an isoquinuclidine
ring, which is a precursor to mearsine (3).17 Also
2-cyclohexenones with formaldehyde and methylamine
produce 6-keto-isoquinuclidine ring systems, as in the
synthesis of dioscorine (2).2 Methylene-bis-urethane,
prepared by Lewis acid-catalyzed Mannich reaction of
urethane with formaldehyde, undergoes cycloaddition
with 1,3-butadiene to form isoquinuclidine carbamate.18
Another widely used approach is via the epoxidation of
the commercially available methyl 3-cyclohexene-1-car-
boxylate using 3-chloroperoxybenzoic acid (m-CPBA),
the product of which subsequently reacts with amines
Compound 6, where the quinoline ring is attached to the
isoquinuclidine through a methyleneamine bridge simi-
lar to that in chloroquine, was prepared from 10 as
shown in Scheme 3. Methylation of 10 with formalde-
hyde and formic acid gave a 90% yield of 16, which on
lithium aluminum hydride reduction gave an 80% yield
of 17. Subsequent conversion of 17 to the target product
6 was achieved by the analogous procedure described in
Scheme 2 for 11–15.
N
H C
3
CH
3
CH
3
R NH C
2
2
N
CH
3
HN
N
HN
or
N
N
Cl
Cl
N
Cl
chloroquine (5)
7
6
Figure 1.
PhH2CO2C
b
N
c
HN
a
N
N
CN
9
CN
10
CO2CH2Ph
8
Scheme 1. Reagents and conditions: (a) benzylchloroformate, NaBH4 , À70 ꢁC, 3 h; (b) benzene, hydroquinone, acrylonitrile; (c) H2/Pd–C, MeOH,
overnight.

M. O. Faruk Khan et al. / Bioorg. Med. Chem. 15 (2007) 3919–3925
3921
NC
H NCH
2
R'RNCH
2
2
Cl
N
N
N
N
N
N
N
HN
a
c
b
+
Cl
Cl
Cl
Cl
CN
10
11
12
13 R=R' = CH
3
14 R = H; R' =C H
2
5
5
15 R = R' = C H
2
Scheme 2. Reagents and conditions: (a) K2CO3 , DMF, 140 ꢁC, 48 h; (b) LAH, rt, 18 h; (c) HCOOH, HCHO (13) or CH3CHO (14, 15).
N
H3C
HN
H3C
H3C
N
N
HN
c
a
b
Cl
N
6
CN
16
CH2NH2
17
CN
10
Scheme 3. Reagents and conditions: (a) HCOOH, HCHO, 60 ꢁ C, 8 h; (b) LAH, rt, 18 h; (c) 4,7-dichloroquinoline, DMF, K2CO3, 140 ꢁC, 24 h.
3. Antimalarial activity
Compound 13 has the isoquinuclidine N attached to the
quinoline ring, thus replacing the secondary amine of
both chloroquine and 6 with a tertiary amine. In 13,
the terminal amine side chain is substituted with the less
bulky methyl groups. Since it is as potent as chloroquine
against the D2 clone and about 7-fold more potent
against W2 clone, compound 13 is suggestive of a prom-
ising antimalarial drug.
All the semirigid analogs of chloroquine synthesized in
the present study (6, 11–15) displayed significant
in vitro antimalarial activity (Table 1). Compound 6 is
the most potent of all these congeners being about 2-fold
more potent against the chloroquine-sensitive D6 clone
and about 30-fold more potent against the chloro-
quine-resistant W2 clone of P. falciparum than chloro-
quine. Its potency is similar to that of artemisinin
against the D2 clone and 0.5-fold less potent against
the W2 clone, which is also significant. Compound 6
possesses the same structural features as chloroquine,
particularly the methyleneamine bridge. However, 6
has the semirigid side chain instead of the open alkyl-
amine side chain of chloroquine. The isoquinuclidine
N atom is substituted with the less bulky methyl group.
Compounds 11, 12, 14 and 15 possess structural features
similar to that of 13 except that they contain terminal ni-
trile, primary amine, ethylamine, and diethyl amine
functions, respectively, furnishing relatively less potent
agents. The reduced potency of nitrile-containing com-
pound 11 may be due to the loss of the basic terminal
amino group of chloroquine required for its antimalarial
potency. Compounds 14 and 15 possess the more bulky
Table 1. In vitro antimalarial activity of isoquinuclidine analogs of chloroquine
Compound
Activity against Plasmodium falciparum
P. falciparum (D6 clone) P. falciparum (W2 clone)
Cytotoxicity
TC50 (ng/ml)
IC50 (ng/ml)
3.8
350
SI
IC50 (ng/ml)
4.0
170
SI
6
11
>1252.3
>13.6
>36.6
>396.7
>17
>1190
>28
NC
NC
NC
NC
NC
NC
—
12
13
130
12
180
18
>26.4
>264.4
>43.2
>23.8
—
14
15
280
310
110
200
115
>15.4
—
—
Chloroquine
Artemisinin
7.0
3.6
1.8
—
—
NC, no cytotoxicity, selectivity index (SI) = IC50 (Vero cells)/IC50 (P. falciparum).

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M. O. Faruk Khan et al. / Bioorg. Med. Chem. 15 (2007) 3919–3925
Table 2. In vitro antileishmanial activity of isoquinuclidine analogs of
chloroquine
The mixture was extracted with ether (3· 50 ml) and
the combined ether layers washed with water (2·
40 ml) and then brine. The ether extract was dried with
anhydrous MgSO4 and evaporated to yield 31 g (96%)
of 8 as a highly unstable oil which was used without fur-
ther purification in the next step as described in the lit-
erature and was not characterized further.27
Compound
Activity against Leishmania donovani
IC50 (lg/ml)
IC90 (lg/ml)
6
11
3.0
16
6.5
33
12
13
4.2
1.9
7.2
3.8
2.8
0.18
20
6.0
29
5.2. 7-Cyano-2-azabicyclo[2.2.2]oct-5-ene-2-carboxylic
acid benzyl ester (9)
14
15
8.0
10
Pentamidine
Amphotericin B
The dihydropyridine derivative 8 was dissolved in ben-
zene (50 ml), and 40 ml of acrylonitrile and 300 mg
hydroquinone added. The mixture was kept at room
temperature for 5 h, then heated at 80 ꢁC for 3 days.
An additional 40 ml of acrylonitrile was added and heat-
ing under reflux continued for an additional 2 days. The
reaction mixture was cooled to room temperature, evap-
orated to dryness, and the residue purified by column
chromatography over silica gel (200–425 mesh) and
eluted with hexane/ethyl acetate (80:20) to yield 15 g of
a mixture of the exo and endo adducts as a colorless
oil that solidified on standing. A portion was purified
to separate exo- and endo-isomers for analytical
purposes.
1.8
ethyl substituent, which might be responsible for its
lower potency. Other stereochemical factors required
for antimalarial potency have not been studied in great
detail since a larger population of analogs would be
required. However, the present study shows that explo-
ration of rigid and semi-rigid congeners can lead to
potentially useful increases in drug properties in this
field. In general, it can be commented that as they
possess similar structural features as chloroquine, they
would be expected to act by a similar mechanism to
chloroquine viz., by interfering with the conversion of
free heme to hemozoine.20–23 The parasite thus accumu-
lates large quantities of heme in the food vacuoles which
lyses membranes, generates reactive oxygen intermedi-
ates, and inhibits many other processes important to
the parasite. The Plasmodium parasites detoxify this
heme by forming the hemozoine or malarial pigment.
However, more detailed studies are needed to define
the mode of action.
1H NMR (400 MHz; CDCl3): exo: 7.35 (5H, m, Ar),
6.35–6.5 (2H, m, 7,8-ethylene), 5.15 (2H, m, CH2-Bz),
4.9–5.1, (1H, m, CH-1), 3.1–3.5, (2H, m, CH2-3), 2.6–
2.85 (2H, m, 2· CH-4 & 6), 1.85 (2H, m, CH2-5). endo:
7.35 (5H, m, Ar), 6.4–6.6 (2H, m, 7,8-ethylene), 5.1 (2H,
m, CH2-Bz), 4.9–5.3, (1H, m, CH-1), 2.8–3.25 (4H, m,
2· CH-4 & 6, CH2-3), 1.6–2.0 (2H, m, CH2-5). (EIMS:
269.4 M+1) 270.3 (M+2).
4. Antileishmanial activity
5.3. 2-Azabicyclo[2.2.2]octane-6-carbonitrile (10)
Most of these compounds also displayed potent in vitro
antileishmanial activity (Table 2). Compound 11, which
contains a terminal nitrile function, is the least potent
with 6 and 13 being the most potent (IC50 3.0 and
1.9 lg/ml, respectively). These potencies are comparable
to that of the standard drug pentamidine (2.8 lg/ml),
but are 10- to 40-fold less potent than amphotericin B
(IC50 0.18 lg/ml). Few examples of antileishmanial
activity of 4-amino- and 8-aminoquinoline derivatives
have been reported,24–26 and none possess any further
detail about the mechanism of action.
To 5 g of the Diels–Alder adduct 9 (endo) dissolved
in methanol was carefully added 500 mg of Pd–C
(5% w/w). The mixture was hydrogenated overnight at
60 psi, and then filtered under vacuum through a short
column packed with silica at the bottom and Celite on
the top. The filtrate was evaporated and the product left
under high vacuum for 1 h to yield 2.7 g (99%) of 10.
The product was stored under argon atmosphere and
used immediately for the next reaction.
1H NMR (400 MHz; CDCl3): 2.6–3.1 (2H, m, 2· CH-1
& 6), 1.4–2.0 (5H, m, CH-4, 2· CH2-3,5), 1.0–1.2 (4H,
m, CH2-7 & 8). EIMS: 137.2 (M+1).
5. Experimental
5.4. 2-(7-Chloro-quinolin-4-yl)-2-aza-bicyclo[2.2.2]octane-
6-carbonitrile (11)
5.1. 1,2-Dihydropyridine-1-carboxylic acid benzyl ester
(8)
Compound 10 (2.5 g) was added to a hot mixture of
DMF (4 ml), 4,7-dichloroquinoline (400 mg), and
K2CO3 (600 mg). The mixture was heated to 140 ꢁC
for 48 h, evaporated to dryness, and the residue parti-
tioned between water (50 ml) and dichloromethane
(50 ml). The dichloromethane layer was separated and
the aqueous layer extracted twice more with dichloro-
methane. The combined dichloromethane extracts were
washed with water and brine, dried over anhydrous
Sodium borohydride (5.7 g, 0.15 mol) was added por-
tionwise to a solution of anhydrous pyridine (12.1 ml,
0.15 mol) in anhydrous methanol (100 ml) at À70 ꢁC
under argon atmosphere. The reaction was cooled to
À75 ꢁC and benzylchloroformate (24.8 ml, 0.15 ml)
added dropwise. The mixture was stirred for 3 h. Ether
and water were added slowly to the reaction mixture
that was then allowed to warm to room temperature.

M. O. Faruk Khan et al. / Bioorg. Med. Chem. 15 (2007) 3919–3925
3923
MgSO4, and evaporated to yield the crude product. Col-
umn chromatography over silica gel (200–425 mesh)
using chloroform/methanol (9:1) yielded 3.9 g of prod-
uct which was used without further purification for the
next step. For analytical purposes a portion of this
was purified by column chromatography over silica gel
(200–425 mesh) using dichloromethane/methanol (9:1)
as solvent.
Using acetaldehyde rather than formaldehyde in the
above method yielded 14 and 15.
1H NMR (400 MHz; CDCl3) (14): 8.4 (1H, m, Ar), 8.0
(1H, m, Ar), 7.9, (1H, m, Ar), 7.3 (1H, m, Ar), 6.3
(1H, m, Ar), 3.0-3.3 (3H, m, CH-1, CH2-3), 1.2–2.3
(12H, m, 2· CH, 5· CH2), 0.9 (3H, t, CH3). EIMS:
332.3 (M+3).
1H NMR (400 MHz; CDCl3): 8.4 (1H, m, Ar), 7.9 (2H,
m, Ar), 7.3, (1H, m, Ar), 6.3 (1H, m, Ar), 3.3–4.2 (3H,
m, CH-1, CH2-3), 1.3–2.5 (8H, m, 2· CH-4 & 6, 3·
CH2-5,7,8). EIMS: 298.3 (M+1).
1H NMR (400 MHz; CDCl3) (15): 8.4 (1H, m, Ar), 7.7–
7.95 (2H, m, Ar), 7.3 (1H, m, Ar), 6.3 (1H, m, Ar), 3.0–
3.3 (3H, m, CH-1, CH2-3), 0.9–2.6 (20H, m, 2· CH, 6·
CH2, 2· CH3). EIMS: 360.3 (M+3).
5.5. [N-(7-Chloro-quinolin-4-yl)]-6-aminomethyl-2-azabi-
cyclo[2.2.2]octane (12)
5.7. 2-Methyl-2-azabicyclo[2.2.2]octane-6-carbonitrile
(16)
To 100 mg (0.34 mmol) of the nitrile 11 in dry THF
(1 ml) was added drop wise 500 ll of 1 M lithium alumi-
num hydride (LAH) solution in ether under argon atmo-
sphere at 0 ꢁC. The reaction mixture was stirred for 18 h
at room temperature, and then quenched by the drop-
wise addition of 0.5 ml of water at 0 ꢁC. The reaction
mixture was extracted with ether (3· 5 ml) and the aque-
ous precipitate extracted once again with 10 ml of ether
under reflux. The combined organic extracts were
washed with brine, dried with anhydrous MgSO4, and
evaporated to yield the crude product which was used
without further purification for the next step. An analyt-
ical grade sample was obtained from a portion of this by
column chromatography over silica gel (200–425 mesh)
using dichloromethane/methanol/ammonia (9:0.9:0.1)
as solvent.
To 880 mg of 10 was added 900 ll of 37% formaldehyde
in water with cooling. Then 1.5 ml of formic acid (95%)
was added dropwise with cooling, the resulting mixture
stirred for an hour at room temperature, and refluxed
for 6 h. The reaction mixture was cooled, 5% NaOH
solution added to raise the pH >11, and extracted with
dichloromethane (3· 10 ml). The organic layers were
combined, washed with brine, dried with MgSO4, and
evaporated to yield 16 (870 mg, 90%). A portion was
purified for analytical purposes and the remainder used
without further purification in the next step.
1H NMR (400 MHz; CDCl3): 3.1 (1H, m, CH-1), 2.6–
2.75 (2H, m, CH2-3), 2.35 (3H, s, N-CH3), 1.85–2.25
(4H, m, 2· CH-4 & 6, CH2-3), 1.3–1.75 (4H, m, 2·
CH2-7 & 8). EIMS: 151.2 (M+1).
1H NMR (400 MHz; CDCl3): 8.4 (1H, m, Ar), 7.65–8.0
(2H, m, Ar), 7.3 (1H, m, Ar), 6.3 (1H, m, Ar), 3.0–3.2
(3H, m, CH-1, CH2-3) 1.3–2.7 (12H, m, 2· CH-4 & 6,
3· CH2-5,7,8, CH2–NH2). EIMS: 302.3 (M+1), 303.3
(M+2), 304.3 (M+3).
5.8. 6-Aminomethyl-2-methyl-2-azabicyclo[2.2.2]octane
(17)
To 750 mg (5 mmol) of 16 in 5 ml of anhydrous THF
was added drop wise 5 ml of 1 M solution of LAH in
ether at 0 ꢁC. The mixture was stirred at room tempera-
ture for 20 h, cooled to 0 ꢁC, and then a mixture of 1 ml
of water and 10 ml of ether was carefully added. The
mixture was stirred overnight and the organic layer
taken off by decantation, washed with brine, and dried
with anhydrous MgSO4. Evaporation of solvent to
afford a yield of 600 mg (80%) of 17 which was used
without further purification in the next step.
5.6. [2-(7-Chloro-quinolin-4-yl)-6-dimethylaminomethyl-
2-azabicyclo[2.2.2]octane (13)
The amine was placed in a 25 ml round bottom flask
and to this were added 100 ll of 37% formaldehyde
solution and 200 ll of formic acid. The mixture was
heated under reflux at 70 ꢁC for 20 h. The reaction
mixture was cooled, and 5% NaOH added until alka-
line whereupon it was extracted with chloroform,
dried over anhydrous MgSO4 and evaporated to dry-
ness to provide the crude product. This product was
then chromatographed over silica gel (200–425 mesh),
eluting with chloroform/methanol (8:2) to wash out
the less polar impurities, and then with chloroform/
methanol/ammonia (8:1.75:0.25) to provide 30 mg of
pure 13.
5.9. (7-Chloro-quinolin-4-yl)-(2-methyl-2-azabicyclo-
[2.2.2]oct-6-ylmethyl)amine (6)
To a hot mixture of DMF (1 ml), 4,7-dichloroquinoline
(400 mg), and K2CO3 (600 mg) was added 300 mg of 17.
The mixture was heated to 140 ꢁC for 24 h, evaporated
to dryness, and then treated with water (10 ml) and
dichloromethane (10 ml). The dichloromethane layer
was separated and the aqueous layer extracted twice
more with dichloromethane. The combined dichloro-
methane extracts were washed with water and brine,
dried with anhydrous MgSO4, and evaporated to yield
the crude product which was purified by column chro-
matography over silica gel (200–425 mesh) using chloro-
form/methanol (9:1) to yield 300 mg of 6.
1H NMR (400 MHz; CDCl3): 8.4 (1H, m, Ar), 7.9–7.7
(2H, m, Ar), 7.3 (1H, m, Ar), 6.3 (1H, m, Ar), 3.0–3.3
(3H, m, CH-1, CH2-3), 2.2 (6H, s, N(CH3)2), 1.9–2.3
(2H, m, overlapped with CH3 peaks, CH2–N), 1.3–
1.85 (8H, m, 2· CH-4 & 6, 3· CH2-5,7,8). EIMS:
332.3 (M+3).

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M. O. Faruk Khan et al. / Bioorg. Med. Chem. 15 (2007) 3919–3925
1H NMR (400 MHz; CDCl3): 9.2 (1 H, br s, NH-Ar),
8.4 (1H, m, Ar), 7.9 (1H, m, Ar), 7.65 (1H, m, Ar), 7.3
(1H, m, Ar), 6.3 (1H, m, Ar), 3.2–3.5 (3H, m, CH-1,
CH2–N–Ar), 2.7 (1H, m, CH-6), 2.4 (3H, s, N-CH3),
1.4–2.3 (9H, m, CH-4, 4· CH2-3,5,7,8). EIMS: 316.3
(M+1).
of the compounds will also be studied in addition to
their activity on hemozoin formation20 to demonstrate
the mechanism of their antimalarial activity.
Acknowledgments
The authors are grateful to the Centers for Disease Con-
trol and Prevention for supporting this research under
Cooperative Agreement U50/CCU423310 and to the
USDA under Cooperative Agreement No. 58-6408-2-
0009 for supporting the antimalarial assays. The authors
also acknowledge John Trott, Mahitha Oruganti, and
Dr. Shabana Khan of The University of Mississippi
National Center for Natural Products Research, for
performing the antimalarial and antileismanial assays.
5.10. Bioassays
5.10.1. In vitro antimalarial assay. The compounds were
assayed for in vitro antimalarial activity against the W2
clone and D6 clone of the pathogenic Plasmodium falci-
parum. The strains of P. falciparum were obtained from
the Division of Experimental Therapeutics, Walter Reed
Army Institute of Research, Washington DC. Strains of
Sierra Leon D6 and Indochina W2 are chloroquine-sen-
sitive and chloroquine-resistant, respectively. The para-
site was grown in type A human RBCs and, two
strains subcultured daily with fresh medium and blood
cells. On the day of the assay, a suspension of infected
blood cells (2% parasitemia and 2% hematocrit) was
prepared using type A human red blood cells. Assays
were performed by standard 96-well flat-bottomed
microplate methods and done in duplicate at different
concentrations. IC50 values were obtained from the dose
curves. Artemisinin and chloroquine were included in
each assay as the drug controls and DMSO was used
as vehicle control. The whole protocol was developed
based on the published method of Makler.28 The cyto-
toxicity assays were also performed in parallel against
the Vero cells and the selectivity index was measured.
A selectivity index of >9 was considered to indicate a
lead for further development of antimalarial drug.
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